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Plasmid Quickpreps

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Plasmid Quickpreps
Reference:  Sambrook,1989 Last updated: 1/27/00 By: Kay Schneitz
  1. grow up single colony in 1.5 ml LB/antibiotic ovn @37°C
  2. pour into tube, spin for 30 sec
  3. decant supernatant and resuspend pellet in 100 ml lysis solution
  4. add 200 ml alkaline SDS, vortex well
  5. incubate for 5 min on ice
  6. add 150 ml high salt solution, vortex well
  7. incubate for ca. 10 min on ice
  8. spin for 10 min
  9. remove 400 ml supernatant and transfer to new tube prefilled with 400 ml isopropanole
  10. vortex well and keep tube on ice for 10 min
  11. spin for 10 min
  12. wash pellet with 70% EtOH
  13. vacuum dry pellet for 5 min and take up in 100 ml 1x TE/20 mg/ml RNAse A
  14. usually 1-2 ml is enough for digest (high-copy plasmid), keep DNA frozen at -20°C
  15. phenolize important preps if to be kept for a longer period of time (more than 4-6 months)





lysis solution:
(freshly prepared)

 7.55 ml H2O
 2    ml 50% glucose
 0.2  ml 0.5 M EDTA
 0.25 ml 1 M Tris-HCl pH 8
10    ml total

alkaline SDS:
(stable for 1 week)

 7.6  ml H2O
 2    ml 5% SDS
 0.4  ml 5 N NaOH
10    ml total

hight salt solution:

3 M NaOAc pH 5.2
(same solution as used in precipitating DNA)



Even if kept at -20° C DNA from this quality will degrade over a period of a year or so when not phenolized carefully (ca. 3 x)! Take care that no interphase remains.


  © Copyright 1997-2000, Kay Schneitz. Last updated 12/31/98; 12:02:17 PM. Send comments to the webmeister.